Methods for recovering peptides from stress protein-peptide complexes

ABSTRACT

Disclosed is a method for inhibiting the proliferation of a tumor in a mammal. The method involves the steps of (a) isolating a stress protein-peptide complex from tumor cells previously removed from the mammal and (b) administering the isolated stress protein-peptide complex back to the mammal in order to stimulate in the mammal an immune response against the tumor from which the complex was isolated. Stress protein-peptide complexes having particular utility in the practice of the instant invention include the Hsp70-peptide, Hsp90-peptide and gp96-peptide complexes.

FIELD OF THE INVENTION

The application relates generally to the field of cancer therapy, inparticular, to the immunotherapy of human cancer.

BACKGROUND OF THE INVENTION

It has been found that inbred mice and rats can be immunizedphrophylactically against tumors derived from mice and rats of the samegenetic background (Gross (1943) Cancer Res. 3: 323-326′; Prehn et al.(1957) J. Natl. Cancer Inst. 18: 769-778; Klein et al. (1960) CancerRes. 20: 1561-1572; Old et al. (1962) Ann NY Acad. Sci. 101: 80-106; forreview, see Srivastava et al. (1988) Immunology Today 9: 78-83). Thesestudies not only showed that mice vaccinated with inactivated cancercells become immunized against subsequent challenges of live cancercells but also demonstrated the existence of tumor-specific antigens.

Further studies revealed that the phenomenon of prophylactically inducedimmunity is tumor-specific. Although mice can be specifically immunizedagainst the tumor cells that were used to immunize them they stillremain sensitive to challenges with other unrelated tumors (Basombrio(1970) Cancer Res. 30: 2458-2462, Globerson et al. (1964) J. Natl.Cancer Inst. 32: 1229-1243). The demonstration of immunogenicity ofcancer cells led to a search for the cancer-derived molecules whichelicit resistance to tumor challenges. The general approach was tofractionate cancer cell-derived proteins and test them individually fortheir ability to immunize mice against the cancers from which thefractions were prepared (see Srivastava et al. (1988) supra; Old (1981)Cancer Res. 41: 361-375). A number of proteins have been identified bythis method, however, a large proportion of these proteins are relatedto a class of proteins known as stress-induced proteins or stressproteins (Lindquist et al. (1988) Annual Rev. Genet. 22: 631-677).Because the stress proteins are among the most highly conserved andabundant proteins in nature, they are unlikely candidates for tumorspecific antigens. Stress proteins have subsequently been shown to noncovalently associate with a variety of peptides thereby to form stressprotein-peptide complexes (Gething et al. (1992) Nature 355: 33-45;Lindquist et al. (1988) supra; Young (1990) Annu. Rev. Immunol. 8:401-420; Flynn et al. 1991) Nature 353: 726-730).

Studies have also shown that stress protein-peptide complexes lose theirimmunogenicity upon treatment with ATP (Udono et al. (1993) J. Exp. Med.178: 1391-1396). This treatment is known to dissociate thestress-protein peptide complex into its stress protein and peptidecomponents. Considering that there are no differences in the structureof stress proteins derived from normal and tumor cells, and that stressproteins bind a wide spectrum of peptides in an ATP dependent manner itappears that the antigenicity of the stress protein-peptide complexresults not from the stress protein per se, but from the peptideassociated with the stress protein.

One of the major conceptual difficulties in cancer immunotherapy hasbeen the possibility that human cancers, like cancers of experimentalanimals, are antigenically distinct. Clearly, there is some recentevidence for existence of common tumor antigens (Kawakami et al. (1992)J. Immunol. 148: 638-643; Darrow et al. (1989) J. Immunol. 142:3329-3334), and this augurs well for prospects of cancer immunotherapy.Nonetheless, in light of the overwhelming evidence from experimental andhuman systems, it is reasonable to assume that at the very least, humantumors would show tremendous antigenic diversity and heterogeneity.

The prospect of identification of the immunogenic antigens of individualtumors from cancer patients (or even of ‘only’ several different typesof immunogenic antigens in case the antigens are shared), is daunting tothe extent of being impractical. Conventional cancer therapies typicallyare based on the isolation and characterization of specific antigenicdeterminants which then may become the target for subsequentimmunotherapies. In addition, although studies have demonstrated thatmammals can be immunized prophylactically against tumors derived frommammals of the same genetic background, heretofore it has not beenappreciated that a mammal harboring a tumor can be therapeuticallyimmunized with a composition derived from its own tumor as a means oftreating a cancer preexisting in the mammal.

Accordingly, it is an object of the instant invention to provide a novelmethod for therapeutically inhibiting proliferation of tumors in amammal. The method described herein does not require the isolation andcharacterization of specific antigenic determinants, and accordinglyprovides a more rapid approach for making and using immunogeniccompositions effective in inhibiting the proliferation of specificpredetermined tumors in mammals.

This and other objects and features of the invention will be apparentfrom the description and claims which follow.

SUMMARY OF THE INVENTION

The observation that stress proteins chaperone the antigenic peptides ofthe cells from which they are derived provides an approach for readilyisolating antigenic peptides for a preselected tumor. Once isolated, thestress protein-peptide complexes are administered back to the animalfrom which they were derived in order to elicit an immune responseagainst a preexisting tumor. Accordingly, this approach circumvents thenecessity of isolating and characterizing specific tumor antigens andenables the artisan to readily prepare immunogenic compositionseffective against a preselected tumor.

In its broadest aspect, the invention provides a method for inhibitingproliferation of a preselected tumor in a mammal. The method comprisesadministering to the mammal undergoing therapy a composition comprisinga pharmaceutically acceptable carrier in combination with a stressprotein-peptide complex. The complex having been isolated from a tumorcell previously excised from the mammal and characterized in that it isoperative to initiate in the mammal an immune response against the tumorcells from which it was derived. The complex subsequently isadministered back to the mammal in an amount sufficient to elicit in themammal an immune response against the tumor cells thereby to inhibitproliferation of any tumor cells still remaining in the mammal.

It is contemplated that this approach may be used n combination withother conventional cancer therapies which include, for example, surgery,radiation therapy and chemotherapy. For example, following surgicalexcision of cancerous tissue the artisan, using the; principlesdescribed herein, may isolate stress protein-peptide complexes from theexcised tissue and administer the complex back to the mammal. Thecomplex subsequently induces a specific immune response against anyremaining tumor cells that were not excised during surgery. The approachis amenable to cancer therapy when the primary tumor has metastasized todifferent locations with the body.

The term “tumor” as used herein, is understood to mean any abnormal oruncontrolled growth of cells which may result in the invasion of normaltissues. It is contemplated also that the term embraces abnormal oruncontrolled cell growths that have metastasized, i.e., abnormal cellsthat have spread from a primary location in the body (i.e., primarytumor) to a secondary location spatially removed from the primary tumor.

The term “stress protein” as used herein, is understood to mean anycellular protein which satisfies the following criteria. It is a proteinwhose intracellular concentration increases when a cell is exposed tostressful stimuli, is capable of binding other proteins or peptides, andis capable of releasing the bound proteins or peptides in the presenceof adenosine triphosphate (ATP) and/or low pH. Stressful stimuliinclude, but are not limited to, heat shock, nutrient deprivation,metabolic disruption, oxygen radicals, and infection with intracellularpathogens.

The first stress proteins to be identified were the heat shock proteins(Hsp's). As their name suggests, Hsp's typically are induced by a cellin response to heat shock. Three major families of mammalian Hsp's havebeen identified to date and include Hsp60, Hsp70 and Hsp90. The numbersreflect the approximate molecular weight of the stress proteins inkilodaltons. The members of each of the families are highly conserved,see for example, Bardwell et al. (1984) Proc. Natl. Acad. Sci. 81:848-852; Hickey et al. (1989) Mol. Cell Biol. 9: 2615-2626; Jindal(1989) Mol. Cell. Biol. 9: 2279-2283, the disclosures of which areincorporated herein by reference. Members of the mammalian Hsp90 familyidentified to date include cytosolic Hsp90 (also known as Hsp83) and theendoplasmic reticulum counterparts Hsp90 (also known as Hsp83), Hsp87,Grp94 (also known as ERp99) and gp96. See for example, Gething et al.(1992) Nature 355: 33-45 the disclosure of which is incorporated hereinby reference. Members of the Hsp70 family identified to date include:cytosolic Hsp70 (also known as p73) and Hsc70 (also known as p72); theendoplasmic reticulum counterpart BiP (also known as Grp78); and themitochondrial counterpart Hsp 70 (also known as Grp75), Gething et al.(1992) supra. To date, members of the mammalian Hsp60 family have onlybeen identified in the mitochondria, Gething et al. (1992) supra.

In addition, it has been discovered that the Hsp-60, Hsp-70 and Hsp-90families are composed of proteins related to the stress proteins inamino acid sequence, for example, having greater than 35% amino acididentity, but whose expression levels are not altered by stressfulstimuli. Accordingly, it is contemplated that the definition of stressprotein, as used herein, embraces other proteins, muteins, analogs, andvariants thereof having at least 35% to 55%, preferably 55% to 75%, andmost preferably 75% to 85% amino acid identity with members of the threefamilies whose expression levels in a cell are stimulated in response tostressful stimuli.

The term “peptide”, as used herein, is understood to mean any amino acidsequence isolated from a mammalian tumor cell in the form of a stressprotein-peptide complex.

The term “immunogenic stress protein-peptide complex”, as used herein,is understood to mean any complex which can be isolated from a mammaliantumor cell and comprises a stress protein non covalently associated witha peptide. The complex is further characterized in that it is operativeto induce in the mammal an immune response against the tumor cells fromwhich the complex was derived.

The term “immune response” is understood to mean any cellular processthat is produced in the mammal following stimulation with an antigen andis directed toward the elimination of the antigen from the mammal. Theimmune response typically is mediated by one or more populations ofcells characterized as being lymphocytic and/or phagocytic in nature.

In a more specific aspect of the invention, the stress protein in thestress protein-peptide complex is selected from the group consisting ofHsp70, Hsp90 and gp96. Stress protein-peptide complexes which includeHsp70-peptide, Hsp90-peptide and gp96-peptide complexes may be isolatedsimultaneously from a batch of tumor cells excised from a mammal. Duringimmunotherapy it is contemplated that one or more of the aforementionedcomplexes may be administered to the mammal in order to stimulate theoptimal immune response against the tumor.

It is contemplated that the method described herein is particularlyuseful in the treatment of human cancer. However, it is contemplatedthat the methods described herein likewise will be useful inimmunotherapy of cancers in other mammals, for example, farm animals(i.e., cattle, horses, goats, sheep and pigs) and household pets (i.e.,cats and dogs).

In another aspect of the invention, it is contemplated that the immuneresponse is effected by means of a T cell cascade, and more specificallyby means of a cytotoxic T cell cascade. The term “cytotoxic T cell”, asused herein, is understood to mean any T lymphocyte expressing the cellsurface glycoprotein marker CD8 that is capable of targeting and lysinga target cell which bears a class I histocompatibility complex on itscell surface and is infected with an intracellular pathogen.

In another aspect of the invention, the stress protein-peptide complexesmay be administered to the mammal in combination with a therapeuticallyactive amount of a cytokine. As used herein, the term “cytokine” ismeant to mean any secreted polypeptide that influences the function ofother cells mediating an immune response. Accordingly, it iscontemplated that the complex can be coadministered with a cytokine toenhance the immune response directed against the tumor. Preferredcytokines include, but are not limited to, interleukin-1α (IL-1α),interleukin-1 (IL-1β), interleukin-2 (IL-2), interleukin-3 (IL-3),interleukin-4 (IL-4), interleukin-5 (IL-4), interleukin-6 (IL-6),interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9),interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12),interferon α (IFNα), interferon β (IFNβ), interferon γ (IFNγ), tumornecrosis factor a (TNF∝), tumor necrosis factor β (TNFβ), granulocytecolony stimulating factor (G-CSF), granulocyte/macrophage colonystimulating factor (GM-CSF), and transforming growth factor β (TGF-β).

The complex may be administered to a mammal when combined with aconventional pharmaceutically acceptable carrier, adjuvant, or excipientusing techniques well known in the art. The dosage and means ofadministration of the family of stress protein-peptide complexesnecessarily will depend upon a variety of factors such as the stabilityof the complex under physiological conditions, the effectiveness of thecomplex at eliciting an immune response, the size and distribution ofthe tumor, and the age, sex and weight of the mammal undergoing therapy.

Typically, the complex should be administered in an amount sufficient toinitiate in the mammal an immune response against the tumor from whichthe complex was derived and in an amount sufficient to inhibitproliferation of the tumor cells in the mammal. The amount of stressprotein-peptide complex administered preferably is in the range of about1-1000 micrograms of complex/kg body weight of themammal/administration, and most preferably about 100-250 micrograms ofcomplex/kg body weight of the mammal/administration. It is contemplatedthat typical dose will be in the range of about 5 to about 20 mg for ahuman subject weighing about 75 kg. In addition, it is contemplated thatthe strength of the immune response may be enhanced by repeatedlyadministering the complex to the individual. The mammal preferablyreceives at least two doses of the stress protein-peptide complex atweekly intervals. If necessary, the immune response may be boosted at alater date by subsequent administration of the complex. It iscontemplated, however, that the optimal dosage and immunization regimenmay be found by routine experimentation by one skilled in the art.

DETAILED DESCRIPTION

The invention is based on the observation that stress protein-peptidecomplexes chaperone antigenic peptides of the cells from which they arederived. Conventional cancer therapies are based upon the isolation ancharacterization of tumor specific antigens which then become the targetfor a specific therapeutic regime. Because of the antigenic diversity ofmammalian cancers the isolation and characterization of specific tumorantigens for each specific tumor can be impractical. The instantinvention thus provides an alternative approach to cancer immunotherapyby obviating the necessity of isolating and characterizing tumorspecific antigens for each tumor being treated.

The invention described herein provides a method for inhibitingproliferation of a preselected tumor in a mammal. The method comprisesisolating or obtaining tumor cells from the mammal undergoing therapy.This is accomplished readily using conventional surgical procedures wellknown in the art. Typically, tumor cells are excised from the mammalduring routine surgical recision of the tumor. The method then involvesisolating stress protein-peptide complexes from the excised tumor cells.This is accomplished using any one of the isolation procedures describedin detail herein below. The stress protein-peptide complexes arecharacterized in that when they are administered back to the mammal theyare capable of initiating a specific immune response against the sametype of tumor cells that they were derived from. Finally, the methodcomprises the step of administering back to the mammal the isolatedstress protein-peptide complex in an amount sufficient to elicit in themammal an immune response against the tumor cells thereby inhibitingproliferation of any tumor cells remaining in the mammal.

It is contemplated that this approach may be used in combination withone or more conventional cancer therapies which include, for example,surgery, radiation therapy and chemotherapy. For example, followingsurgical excision of cancerous tissue the artisan, using the principlesdescribed herein, may isolate stress protein-peptide complexes from theexcised tissue and administer the complex back to the mammal. Thecomplex then induces in the mammal a specific immune response againstany tumor cells that were not removed during surgery. Alternatively, themethod described herein provides a novel approach for treating cancerwhen the primary tumor has metastasized to multiple locations with thebody. For example, when the cancer has metastasized, making surgicalintervention impractical, a stress protein-peptide complex may be usedeither alone or in combination with another standard chemotherapeuticagent in the treatment of the cancer.

It is contemplated that the invention has particular utility in theimmunotherapy of human cancer, however, it is appreciated that themethodologies described herein may be applied to the treatment ofcancers occurring in, for example, farm animals (i.e., cattle, horses,sheep, goats and pigs) and household pets (i.e., cats and dogs).

The main advantage this approach has over conventional methodologies isthat it is not necessary to isolate and characterize the tumor specificantigen for each tumor. Once the stress protein-peptide complex has beenisolated it is simply administered back the mammal without furthercharacterization. Since the procedures for isolating the immunogeniccomplexes are routine and well known in the art, the artisan may rapidlyand routinely prepare a specific immunogenic composition “tailor-made”for each individual being treated.

Another advantage of the instant method over previous methodologies isthat the administration of purified stress protein-peptide complexesback to the individual from which they were derived eliminates the riskof inoculating the mammal undergoing therapy with potentiallytransforming agents (i.e., transforming DNA) and/or immunosuppressiveagents which can be an issue when the complex is present in abiochemically undefined tumor or tumor extract. In addition, stressprotein-peptide complexes can induce significant tumor immunity in theabsence of adjuvants. Accordingly, while adjuvants may further enhancethe immunotherapeutic properties of the complex, their availability isnot a pre-condition for inducing a significant immune response.

It is contemplated that this method can be used in the treatment of avariety of tumors, for example, tumors that are nesenchymal in origin(sarcomas) i.e., fibrosarcomas; myxosarcomas; liposarcomas;chondrosarcomas; osteogenic sarcomas; angiosarcomas; endotheliosarcomas;lymphangiosarcomas; synoviosarcomas; mesotheliosarcomas; Ewing's tumors;myelogenous leukemias; monocytic leukemias; malignant lymphomas;lymphocytic leukemias; plasmacytomas; leiomyosarcomas andrhabdomyosarcoma.

In addition, it is contemplated that this method can be used in thetreatment of tumors that are epithelial in origin (carcinomas) i.e.,squamous cell or epidermal carcinomas; basal cell carcinomas; sweatgland carcinomas; sebaceous gland carcinomas; adenocarcinomas; papillarycarcinomas; papillary adenocarcinomas; cystadenocarcinomas; medullarycarcinomas; undifferentiated carcinomas (simplex carcinomas);bronchogenic carcinomas; bronchial carcinomas; melanocarcinomas; renalcell carcinomas; hepatocellular carcinomas; bile duct carcinomas;papillary carcinomas; transitional cell carcinomas; squamous cellcarcinomas; choriocarcinomas; seminomas; embryonal carcinomas malignantteratomas and teratocarcinomas. Generic methodologies useful in thepreparation of compositions effective at inducing an immune responseagainst these tumors are discussed in detail herein below.

Although not wishing to be bound by theory, it is contemplated that thestress protein-peptide complexes stimulate an immune response againstthe tumor cells from which they are derived by means of a T cellcascade. Previous experiments have demonstrated that mice immunizedprophylactically with stress protein-peptide preparations derived from atumor originating in the same strain of mouse or rat developimmunological resistance to the tumor from which it was isolated. Themice, however, fail to develop immunity against antigenically distincttumors. Furthermore, stress protein-peptide complexes derived fromnormal tissues do not elicit resistance to any tumors tested. See forexample, Srivastava et al. (1984) Int. J. Cancer 33: 417; Srivastava etal. (1986) Proc. Natl. Acad. Sci. USA 83: 3407; Palladino et al. (1987)Cancer Res. 47: 5074; Feldweg et al. (1993) J. Cell Biochem. Suppl.17D:108 (Abst.); Udono et al. (1993) J. Cell. Biochem. Suppl. 17D:113and Udono (1993) J. Exp. Med. 178: 1391-1396, the disclosures of whichare incorporated herein by reference. Recently, it has been establishedprophylactic immunity typically is mediated by means of a T cellcascade, more specifically by means of a cytotoxic T cell cascade. Seefor example, Blachere et al. (1993) J. Immunother. 14: 352-356, thedisclosure of which is incorporated by reference herein. Accordingly, itis contemplated that the stress-protein complexes may also mediate theireffect therapeutically by a similar mechanism; specifically, via acytotoxic T cell cascade.

It is contemplated that the stress protein-peptide complexes typicallywill be isolated directly from tumor tissue excised from the mammalbeing treated. Under certain conditions, however, the amount of tumortissue available for isolation of the complex may be limiting.Accordingly, it is contemplated that the excised tumor tissue may beproliferated using techniques well known in the art prior to theisolation of the stress protein-peptide complexes. For example, theexcised tumor tissue may be proliferated either in vivo, for example, bytransfecting a nude mouse with a sample of the tumor tissue, or invitro, for example, by serially passaging the tumor cells in culture.The proliferated tumor tissue subsequently can be harvested and used asa starting material for the isolation of the stress protein-peptidecomplex.

Stress proteins useful in the practice of the instant invention may bedefined as any cellular protein that satisfies the following criteria.It is a protein whose intracellular concentration increases when a cellis exposed to a stressful stimuli, is capable of binding other proteinsor peptides, and is capable of releasing the bound proteins or peptidesin the presence of adenosine triphosphate (ATP) or low pH.

The first stress proteins to be identified were the Hsp's which aresynthesized in a cell in response to heat shock. To date, three majorfamilies of mammalian Hsp's have been identified and include Hsp60,Hsp70 and Hsp90 where the numbers reflect the approximate molecularweight of the stress proteins in kilodaltons. Many members of thesefamilies were found subsequently to be induced in response to otherstressful stimuli including, but not limited to, nutrient deprivation,metabolic disruption, oxygen radicals, and infection with intracellularpathogens. See for example: Welch (May 1993) Scientific American 56-64;Young (1990) supra; Craig (1993) Science 260: 1902-1903; Gething et al(1992) supra; and Lindquist et al. (1988) supra, the disclosures ofwhich are incorporated herein by reference. It is contemplated thatmammalian stress proteins belonging to all three families may be usefulin the practice of the instant invention.

The major stress proteins accumulate to very high levels in stressedcells but occur at low to moderate levels in cells that have not beenstressed. For example, the highly inducible mammalian Hsp70 is hardlydetectable at normal temperatures but becomes one of the most activelysynthesized proteins in the cell upon heat shock (Welch et al. (1985),J. Cell. Biol. 101: 1198-1211). In contrast, Hsp90 and Hsp60 proteinsare abundant at normal temperatures in most, but not all, mammaliancells and are further induced by heat (Lai et al. (1984), Mol. Cell.Biol. 4: 2802-10; van Bergen en Henegouwen et al. (1987), Genes Dev., 1:525-31).

Members of the mammalian Hsp90 family identified to date includecytosolic Hsp90 (also known as Hsp83) and the endoplasmic reticulumcounterparts Hsp90 (also known as Hsp83), Hsp87, Grp94 (also known asERp99) and gp96 (Gething et al. (1992) supra). Members of the Hsp70family identified to date include: cytosolic Hsp70 (also known as p73)and Hsc70 (also known as p72), the endoplasmic reticulum counterpart BiP(also known as Grp78) and the mitochondrial counterpart Hsp 70 (alsoknown as Grp75), Gething et al. (1992) supra. To date, members of themammalian Hsp60 family have only been identified in the mitochondria,Gething et al. (1992) supra.

Stress proteins are among the most highly conserved proteins inexistence. For example, DnaK, the Hsp70 from E. coli has about 50% aminoacid sequence identity with Hsp70 proteins from eukaryotes (Bardwell etal. (1984) Proc. Natl. Acad. Sci. 81: 848-852). The Hsp60 and Hsp90families similarly exhibit high levels of intrafamilial conservation(Hickey et al. (1989) Mol. Cell Biol. 9: 2615-2626; Jindal (1989) Mol.Cell. Biol. 9: 2279-2283). In addition, it has been discovered that theHsp60, Hsp70 and Hsp90 families are composed of proteins that arerelated to the stress proteins in sequence, for example, having greaterthan 35% amino acid identity, but whose expression levels are notaltered by stress. Therefore it is contemplated that the definition ofstress protein, as used herein, embraces other proteins, muteins,analogs, and variants thereof having at least 35% to 55%, preferably 55%to 75%, and most preferably 75% to 85% amino acid identity with membersof the three families whose expression levels in a cell are enhanced inresponse to a stressful stimulus.

The immunogenic stress protein-peptide complexes of the invention mayinclude any complex containing a stress protein non covalentlyassociated with a peptide that is capable of inducing an immune responsein a mammal. Preferred complexes include, but are not limited to,Hsp70-peptide, Hsp90-peptide and gp96-peptide complexes. For example,the mammalian stress protein gp96 which is the endoplasmic reticulumcounterpart of the cytosolic Hsp90 may be used in the practice of theinstant invention.

Typical procedures for isolating stress protein-peptide complexes usefulin the practice of the instant invention are set forth in detail below.

Purification of Hsp70-Peptide Complexes.

The purification of Hsp70-peptide complexes has been describedpreviously, see for example, Udono et al. (1993) supra.

Initially, tumor cells are suspended in 3 volumes of 1× Lysis bufferconsisting of 5 mM sodium phosphate buffer (pH7), 150 mM NaCl, 2 mMCaCl₂, 2 mM MgCl₂ and 1 mM phenyl methyl sulfonyl fluoride (PMSF). Then,the pellet is sonicated, on ice, until >99% cells are lysed asdetermined by microscopic examination. As an alternative to sonication,the cells may be lysed by mechanical shearing and in this approach thecells typically are resuspended in 30 mM sodium bicarbonate pH 7.5, 1 mMPMSF, incubated on ice for 20 min and then homogenized in a douncehomogenizer until >95% cells are lysed.

Then the lysate is centrifuged at 1000 g for 10 minutes to removeunbroken cells, nuclei and other cellular debris. The resultingsupernatant is recentrifuged at 100,000 g for 90 minutes, thesupernatant harvested and then mixed with Con A Sepharose equilibratedwith phosphate buffered saline (PBS) containing 2 mM Ca²⁺ and 2 mM Mg²⁺.When the cells are lysed by mechanical shearing the supernatant isdiluted with an equal volume of 2× Lysis buffer prior to mixing with ConA Sepharose. The supernatant is then allowed to bind to the Con ASepharose for 2-3 hours at 4° C. The material that fails to bind isharvested and dialyzed for 36 hours (three times, 100 volumes each time)against 10 mM Tris-Acetate pH 7.5, 0.1 mM EDTA, 10 mM NaCl, 1 mM PMSF.Then the dialyzate is centrifuged at 17,000 rpm (Sorvall SS34 rotor) for20 min. Then the resulting supernatant is harvested and applied to aMono Q FPLC column equilibrated in 20 mM Tris-Acetate pH 7.5, 20 mMNaCl, 0.1 mM EDTA and 15 mM 2-mercaptoethanol. The column is thendeveloped with a 20 mM to 500 mM NaCl gradient and the eluted fractionsfractionated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and characterized by immunoblotting using anappropriate anti-Hsp70 antibody (such as from clone N27F3-4, fromStressGen).

Fractions strongly immunoreactive with the anti-Hsp70 antibody arepooled and the Hsp70-peptide complexes precipitated with ammoniumsulfate; specifically with a 50%-70% ammonium sulfate cut. The resultingprecipitate is then harvested by centrifugation at 17,000 rpm (SS34Sorvall rotor) and washed with 70% ammonium sulfate. The washedprecipitate is then solubilized and any residual ammonium sulfateremoved by gel filtration on a Sephadex^(R) G25 column (Pharmacia).

The Hsp70-peptide complex can be purified to apparent homogeneity usingthis method. Typically 1 mg of Hsp70-peptide complex can be purifiedfrom 1 g of cells/tissue.

Purification of Hsp90-Peptide Complexes.

Initially, tumor cells are suspended in 3 volumes of 1× Lysis bufferconsisting of 5 mM sodium phosphate buffer (pH7), 150M NaCl, 2 mM CaCl₂,2 mM MgCl₂ and 1 mM phenyl methyl sulfonyl fluoride (PMSF). Then, thepellet is sonicated, on ice, until >99% cells are lysed as determined bymicroscopic examination As an alternative to sonication, the cells maybe lysed by mechanical shearing and in this approach the cells typicallyare resuspended in 30 mM sodium bicarbonate pH 7.5, 1 mM PMSF, incubatedon ice for 20 min and then homogenized in a dounce homogenizeruntil >95% cells are lysed.

Then the lysate is centrifuged at 1000 g for 10 minutes to removeunbroken cells, nuclei and other cellular debris. The resultingsupernatant is recentrifuged at 100,000 g for 90 minutes, thesupernatant harvested and then mixed with Con A Sepharose equilibratedwith PBS containing 2 mM Ca²⁺ and 2 mM Mg²⁺. When the cells are lysed bymechanical shearing the supernatant is diluted with an equal volume of2× Lysis buffer prior to mixing with Con A Sepharose. The supernatant isthen allowed to bind to the Con A Sepharose for 2-3 hours at 4° C. Thematerial that fails to bind is harvested and dialyzed for 36 hours(three times, 100 volumes each time) against 10 mM Tris-Acetate pH 7.5,0.1 mM EDTA, 10 mM NaCl, 1 mM PMSF. Then the dialyzate is centrifuged at17,000 rpm (Sorvall SS34 rotor) for 20 min. Then the resultingsupernatant is harvested and applied to a Mono Q FPLC columnequilibrated equilibrated with lysis buffer. The proteins are theneluted with a a salt gradient of 200 mM to 600 mM NaCl.

The eluted fractions are fractionated by SDS-PAGE and fractionscontaining the Hsp90-peptide complexes identified by immunoblottingusing a anti-Hsp90 antibody such as 3G3 (Affinity Bioreagents).Hsp90-peptide complexes can be purified to apparent homogeneity usingthis procedure. Typically, 150-200 μg of Hsp90-peptide complex can bepurified from 1 g of cells/tissue.

Purification of gp96-Peptide Complexes.

Initially, tumor cells are suspended in 3 volumes of 1× Lysis bufferconsisting of 5 mM sodium phosphate buffer (pH7), 150 mM NaCl, 2 mMCaCl₂, 2 mM MgCl₂ and 1 mM phenyl methyl sulfonyl fluoride (PMSF). Then,the pellet is sonicated, on ice, until >99% cells are lysed asdetermined by microscopic examination. As an alternative to sonication,the cells may be lysed by mechanical shearing and in this approach thecells typically are resuspended in 30 mM sodium bicarbonate pH 7.5, 1 mMPMSF, incubated on ice for 20 min and then homogenized in a douncehomogenizer until >95% cells are lysed.

Then the lysate is centrifuged at 1000 g for 10 minutes to removeunbroken cells, nuclei and other cellular debris. The resultingsupernatant is recentrifuged at 100,000 g for 90 minutes, thesupernatant harvested and mixed with Con A Sepharose slurry equilibratedwith PBS containing 2 mM Ca²⁺ and 2 mM Mg²⁺. When the cells are lysed bymechanical shearing the supernatant is diluted with an equal volume of2× Lysis buffer prior to mixing with Con A Sepharose. The supernatant isthen allowed to bind to the Con A Sepharose for 2-3 hours at 4° C. Theslurry is then packed into a column and washed with 1× lysis bufferuntil the OD₂₈₀ drops to baseline. Then the column is washed with ½column bed volume of 10% α-methyl mannoside (α-MM), the column sealedwith parafilm and incubated at 37° C. for 15 min. The column is thencooled to room temperature, the parafilm removed from the bottom of thecolumn, and five column volumes of a α-MM is applied to the column. Theeluate is then fractionated and characterized by SDS-PAGE. Typically,the resulting gp96-peptide complex is about 60 to 95% pure dependingupon the cell type and the tissue to lysis buffer ratio used.

If further purification is required, the sample can be applied to a MonoQ FPLC column equilibrated with a buffer containing 5 mM sodiumphosphate, pH7. The proteins are then eluted from the column with a 0-1MNaCl gradient. The gp96 fraction elutes between 400 mM and 550 mM NaCl.

As an alternative procedure, the gp96 fraction isolated from the 100,000g pellet can be resuspended in 5 volumes of PBS containing 1% sodiumdeoxycholate (without Ca²⁺ and Mg²⁺) and incubated on ice for 1 h. Theresulting suspension is centrifuged for 30 min at 20,000 g and theresulting supernatant harvested and dialyzed against several changes ofPBS (without Ca²⁺ and Mg²⁺) to remove the detergent. The resultingdialysate is centrifuged for 90 min at 100,000 g and the supernatantpurified further. Then calcium and magnesium are both added to thesupernatant to give final concentrations of 2 mM. Then the sample isapplied to a Mono Q HPLC column equilibrated with a buffer containing 5mM sodium phosphate, pH7 and the proteins eluted with a 0-1M NaClgradient. The gp96 fraction elutes between 400 mM and 550 mM NaCl. Thegp96-peptide complexes can be purified to apparent homogeneity usingthis procedure. Typically about 10-20 μg of gp96 can be isolated from 1g cells/tissue using this method.

Formulation and Administration of the Complexes.

Once stress protein-peptide complexes have been purified from theexcised tumor they are administered back to the mammal undergoingtherapy in order to stimulate in the mammal an immune response againsttumor cells from which the complex was derived. The stressprotein-peptide complexes of the invention may either be stored orprepared for administration by mixing with physiologically acceptablecarriers, excipients, or stabilizers. These materials should benon-toxic to the intended recipient at dosages and concentrationsemployed.

When the complex is water soluble it may be formulated in an appropriatebuffer, for example PBS (5 mM sodium phosphate, 150 mM NaCl, pH7.1) orother physiologically compatible solutions. Alternatively, if theresulting complex has poor solubility in aqueous solvents then it may beformulated with a non-ionic surfactant such as Tween, or polyethyleneglycol.

Useful solutions for oral or parenteral administration may be preparedby any of the methods well known in the pharmaceutical art, described,for example, in Remington's Pharmaceutical Sciences, (Gennaro, A., ed.),Mack Pub., 1990. Formulations may include, for example, polyalkyleneglycols such as polyethylene glycol, oils of vegetable origin,hydrogenated naphthalenes, and the like. Formulations for directadministration, in particular, may include glycerol and othercompositions of high viscosity. Biocompatible, preferably bioresorbablepolymers, including, for example, hyaluronic acid, collagen, tricalciumphosphate, polybutyrate, polylactide, polyglycolide andlactide/glycolide copolymers, may be useful excipients to control therelease of the stress protein-peptide complexes in vivo.

Formulations for inhalation may contain as excipients, for example,lactose. Aqueous solutions may contain, for example,polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate. Oilysolutions may be useful administration in the form of nasal drops. Gelsmay be applied topically intranasally.

The compounds provided herein can be formulated into pharmaceuticalcompositions by admixture with pharmaceutically acceptable nontoxicexcipients and carriers. In addition the formulations may optionallycontain one or more adjuvants. Preferred adjuvants include, but are notlimited to, pluronic tri-block copolymers, muramyl dipeptide and itsderivatives, detoxified endotoxin, saponin and its derivatives such asQS-21 and liposomes. The present invention further envisages sustainedrelease formulations in which the complex is released over an extendedperiod of time.

The mode of administration of the family of stress protein-peptidecomplexes prepared in accordance with the invention will necessarilydepend upon the stability of the complex under physiological conditions,and the size and distribution of the tumor within the mammal beingtreated. The preferred dosage of complex to be administered also islikely to depend on such variables as the size and distribution of thetumor, the age, sex and weight of the intended recipient, the overallhealth status of the particular recipient, the relative biologicalefficacy of the complex, the formulation for the complex, the presenceand types of excipients in the formulation, and the route ofadministration.

In general terms, the compounds of this invention may be provided in anaqueous physiological buffer solution containing about 0.001 to 10% w/vcompound for parenteral administration. Preferred dosages range fromabout 1 to about 1000 micrograms of complex/kg body weight ofrecipient/administration and most preferably range from about 100 toabout 250 micrograms of complex/kg body weight ofrecipient/administration. In particular, it is contemplated that atypical dose will range from about 5 mg to about 20 mg for a humansubject weighing about 75 kg. These quantities, however, may varyaccording to the adjuvant coadministered with the complex.

The complex preferably comprises part of an aqueous solution which maybe administered using standard procedures, for example, intravenously,subcutaneously, intramuscularly, intraorbitally, ophthalmically,intraventricularly, intracranially, intracapsularly, intraspinally,intracisternally, intraperitoneally, buccal, rectally, vaginally,intranasally or by aerosol administration. The aqueous solutionpreferably is physiologically acceptable so that in addition to deliveryof the desired complex to the mammal, the solution does not otherwiseadversely affect the mammal's electrolyte and/or volume balance. Theaqueous medium for the complex thus may comprise normal hysiologicsaline (0.9% NaCl, 0.15M), pH 7-7.4 or other pharmaceutically acceptablesalts thereof.

Preferably the recipient should be vaccinated three times at two weekintervals. If necessary, the responses may be boosted at a later date bysubsequent administration of the complex. It is contemplated that theoptimal dosage and vaccination schedule may be determinedempirically-for each stress protein-peptide complex using techniqueswell known in the art.

Various cytokines, antibiotics, and other bioactive agents also may becoadministered with the stress protein-peptide complexes. For example,various known cytokines, i.e., interleukin-1α (IL-1α), interleukin-1β(IL-1β), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4(IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7(IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10(IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interferon α(IFNα), interferon β (IFNβ), interferon γ (IFNγ), tumor necrosis factora (TNF∝), tumor necrosis factor β (TNFβ), granulocyte colony stimulatingfactor (G-CSF), granulocyte/macrophage colony stimulating factor(GM-CSF), and transforming growth factor β (TGF-β) may be coadministeredwith the complexes in order to maximize the physiological response.However, it is anticipated that other but as yet undiscovered cytokinesmay be effective in the invention. In addition, conventional antibioticsmay be coadministered with the stress protein-peptide complex. Thechoice of suitable antibiotics will however be dependent upon thedisease in question.

EXAMPLE I

In this example, C57BL/6 and C3H mice approximately 100 g in weight, arepurchased from Jackson Laboratories, Bar Harbor, Me. Malignant tumorcells are then injected subcutaneously into mice in order to induceexperimental tumors in the mice. Specifically, malignant spindle cellcarcinoma 6139 cells are injected subcutaneously into the C3H mice,malignant mouse Lewis lung carcinoma cells are injected subcutaneouslyinto C57BL/6 mice and malignant mouse B16 melanoma cells are injectedsubcutaneously into C57BL/6 mice.

When the tumors have grown to a size such that they are both visible andpalpable, a sample of the tumor tissue is excised. As a control, normalnon malignant tissue is excised from some mice bearing the experimentaltumors.

Then gp96-peptide, Hsp90-peptide and Hsp70-peptide complexes areisolated from both the excised normal and tumor derived tissues usingthe methods described hereinabove. Once isolated, the complexes arecombined with PBS and administered back to the mice from which thecomplexes were derived. Usually 6 mice are tested in each experiment.The experiments are performed using the schedule set forth below:Experiment Composition administered back to mice 1 gp96-peptide 2Hsp70-peptide 3 Hsp90-peptide 4 gp96-peptide and Hsp70-peptide 5gp96-peptide and Hsp90-peptide 6 Hsp70-peptide and Hsp90-peptide 7Hsp70-peptide, Hsp90-peptide and gp96-peptide 8 buffer alone

In one series of experiments the complexes are isolated from tumor cellswhereas in a second series the complexes are isolated from normal cells.The mice are inoculated three times at weekly intervals with 20micrograms (total weight) of the preselected complex(es). Duringtherapy, the size of each tumor is measured daily. After 4 weeks themice are sacrificed and the development of the tumor examinedhistologically. In addition, the sacrificed mice are examined for thepresence or absence of metastasis.

It is expected that the tumors in mice treated with complexes derivedfrom normal tissue will continue to grow and metastasize. In contrast,it is expected that the tumors in the mice treated with the complexesderived from the tumor tissue will be exhibit slower growth than thetumors in the control animals, and in some cases, it is expected thatthe tumor mass may get smaller and the tumor exhibit remission duringtherapy.

Other Embodiments

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The presentembodiments are therefore to be considered in all respects asillustrative and not restrictive, the scope of the invention beingindicated by the appended claims rather than by the foregoingdescription, and all changes which come within the meaning and range ofequivalency of the claims are therefore intended to be embraced therein.

1. A method for inhibiting proliferation of a tumor in a mammal, themethod comprising: administering to the mammal harboring the tumor acomposition comprising, (a) an immunogenic stress protein-peptidecomplex isolated from a cell derived from the tumor, said complex beingoperative to initiate in the mammal an immune response against saidtumor, and (b) a pharmaceutically acceptable carrier, in an amountsufficient to elicit in the mammal an immune response against the tumorthereby inhibiting proliferation of the tumor. 2-18. (canceled)
 19. Amethod of preparing a composition comprising a recovered population ofpeptides in admixture with a pharmaceutically acceptable nontoxiccarrier, wherein said method comprises the steps of: (a) purifying apopulation of stress protein-peptide complexes from mammalian tumorcells, wherein the stress protein is noncovalently associated with thepeptide in said complexes; (b) releasing the peptides from saidpopulation of complexes to produce a released population of peptides;(c) recovering the released population of peptides; and (d) combiningsaid released population of peptides with a pharmaceutically acceptablenontoxic carrier.
 20. The method of claim 19, wherein the peptides arereleased from said population of complexes by a method comprisingplacing said population of complexes in the presence of adenosinetriphosphate.
 21. The method of claim 19, wherein said mammalian tumorcells are human cells.
 22. The method of claim 19, wherein saidmammalian tumor cells are from a tumor selected from the groupconsisting of melanocarcinoma, hepatocarcinoma, and renal cellcarcinoma.
 23. The method of claim 19, wherein said tumor cells are froma metastasis.
 24. The method of claim 19, wherein said tumor cells havebeen proliferated in vivo.
 25. The method of claim 19, wherein saidtumor cells have been proliferated in vitro.
 26. The method of claim 19,wherein the stress protein is a member of a stress protein familyselected from the group consisting of hsp60, hsp70 and hsp90.
 27. Themethod of claim 19, wherein the stress protein is hsp70.
 28. The methodof claim 19, wherein the stress protein is hsp90.
 29. The method ofclaim 19, wherein the stress protein is gp96.
 30. The method of claim19, further comprising combining said released population of peptideswith an adjuvant.
 31. The method of claim 30, wherein the adjuvant isselected from the group consisting of a pluronic tri-block copolymer,muramyl dipeptide, detoxified endotoxin, saponin, QS-21, and liposome.